Helicobacter pylori (H. pylori) is a curved or spiral gram-negative bacterium which infects the gastric mucosal and is responsible for most peptic ulcer disease (PUD). Since this first isolation, Until recently, ulcers and other forms of dyspepsia were thought to be related to stress levels or eating habits. Recently, the medical community has confirmed that H. pylori is the causative agent for certain forms of gastric distress, including ulcers and gastric cancer. Eradication of H. pylori promotes healing of ulcer and greatly reduces the incidences of cancer and PUD.
H. pylori causes most gastric and duodenal ulcers, as well as peptic ulcer disease (PUD). The linkage of H. pylori and PUD was first discovered and published by Australian physicians Warren and Marshall in 1984 (Lancet I: 1311-1344). The H. pylori infection is now accepted as the most common cause of gastritis, and is etiologically involved in gastric ulcer, duodenal ulcer, gastric adenocarcinoma and primary B-cell lymphoma.
It has been proven that PUD is curable and rather easily. The cause of most PUD is infection with H. pylori. However, H. pylori infection is not routinely diagnosed, possibly because methods of testing for H. pylori infection are not satisfactory to physicians, especially the primary care physicians (i.e. invasive biopsy test). Therefore, primary physicians have tended to treat symptomatic patients with antisecretory agents.
Physicians need a simple, accurate and inexpensive diagnostic test for H. pylori infection so that they know when to treat patients and when to refer the patients to a gastroenterologist. However, the currently available H. pylori tests, which can be categorized as invasive tests and noninvasive tests, are not completely satisfactory.
The invasive tests require the use of endoscope followed by biopsy procedure. The tissue samples taken by the biopsy procedure can then be analyzed by culture, histology, or rapid urease testing.
Although culturing of the biopsy specimens provides the most reliable results for H pylori testing, the reports of successful rates in a good laboratory are only between 70% and 80% (Han, S. W., et al., Eur. J. Clin. Microbiol. Infect. Dis. (1995), 14:349-352). Histological examination of special stained biopsy specimens can provide the direct evidence of acute or chronic inflammatory mucosal cells and lesions. However, it requires the collaborations of both an endoscopist and a pathologist (Genta, R. M., et al., Hum. Pathol. (1994), 25:221-226). Rapid urease tests detect the rise in pH from ammonia produced by H. pylori urease, which splits urea into ammonia and carbon dioxide. However, it requires a high density of bacteria and anything that reduces the bacterial load may produce a false-negative (Cutler, A. F., Am. J. Med. (1996), 100:35S-39S).
A number of noninvasive tests have been developed to detect the presence of H. pylori infection since 1990. For example, the Urea Breath Testing is based on the urease activity of the organism, which splits urea labeled with 13C or 14C into nonradioactive 13CO2 or radioactive 14CO2. The urea breath test is widely recommended for confirming eradication of H. pylori 4 weeks after therapy.
U.S. Pat. Nos. 5,716,791, 5,871,942, and 5,932,430 disclose immunoassays for detecting H. pylori antigens in stool specimens using a polyclonal antibody which is obtained from sensitizing animal with H. pylori cells (i.e., ATCC strain 43504). The antibody is purified by DEAE (diethylaminoethyl cellulose) column. Although the stool antigen test is reported to be satisfactory, the collection and process of the stool specimens are found to be difficult and unpleasant. Many patients are unwilling to provide stool samples to physician due to offensive odor and lack convenient collection device.
Serologic testing of serum H. pylori antibodies using ELISA is another widely used test. Examples of the latter techniques can be found in a U.S. Pat. No. 5,262,156 and EP Pat. No. 0 329 570. There have been several major antigens identified and used in immunoassays in the detection of H. pylori antibodies. However, these assays have not exhibited the specificity and sensitivity that are desired in serodiagnosis. (Newell, D. G., et al., Serodian. Immunother. Infec. Dis., (1989), 3:1-6). One of the problems derives from cross-reactivity. That is because the dominant antigens in H. pylori (e.g., the putative flagellar protein which has a molecular weight of 60 Da) are not specific to H. pylori. Some of these antigens can be found in other bacteria such as C. jejuni and C. coli. A second problem that has been encountered in designing immunoassays for H. pylori is strain variation. Substantial differences in the antigens have been observed in different strains of H. pylori. These problems preclude designing an assay around the use of a single antigen. One approach that has been taken to improving the specificity and selectivity of antibody immunoassays for H. pylori has been to use a mixture of antigens from different H. pylori strains which mixture is enriched with certain antigen fragments. One ELISA which detects H. pylori antibodies in blood sera is commercially available. This assay uses a bacterial whole cell lysate as the antigen.
There are other disadvantages of using an ELISA which employs antigens to detect the presence of H. pylori antibodies in serum. In particular, the antibody titer in human sera remains high for a prolonged time (in some cases as much as twelve months) after the infection has been treated. Consequently, a positive test using this ELISA does not necessarily mean that the patient is currently infected and requires treatment for H. pylori infection. When confronted with a positive ELISA, treating physicians often order a gastric biopsy to confirm the presence of the bacteria before initiating antibiotic therapy. Therefore, the antigen-based ELISA does not eliminate the need for the invasive procedure.
It is therefore the object of the present invention to design a noninvasive and highly accurate diagnostic test for H. pylori infection. During the course of the investigation, H. pylori antigens in blood are discovered, which are in the forms of DNA or fragments thereof, or proteins/peptides or other antigenic components thereof, exist in blood, including whole blood, plasma and serum. Special methods for detecting these H. pylori antigens are thus designed to provide evidence that antigenic fragments of H. pylori are existed in blood. These methods include, but not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR) and DNA hybridization for detecting nucleic acid fragments of H. pylori, using primers or oligonucleotides specific for H. pylori and/or DNA probes derived from H. pylori strains. Additionally, immunoassays and immunoblotting are also developed for detecting protein/peptide or any antigenic components of H. pylori, using an affinity purified antibody against H. pylori. 
There has been no report with regard to the existence of H. pylori antigens in blood. The present invention is the first to prove that H. pylori antigens not only exist in blood, but can be detected by the methods presented in the following sections